The present invention provides plant retroelements and methods related to plant retroelements. The invention involves techniques from the fields of: molecular biology, virology, genetics, bioinformatics, and, to a lesser extent, other related fields.
The eukaryotic retrotransposons are divided into two distinct classes of elements based on their structure: the long terminal repeat (LTR) retrotransposons and the LINE-like or non LTR elements. Doolittle et al. (1989) Quart. Rev. Biol. 64: 1-30; xiong and Eickbush (1990) EMBO J 9: 3353-3362. These element classes are related by the fact that each must undergo reverse transcription of an RNA intermediate to replicate, and each generally encodes its own reverse transcriptase. The LTR retrotransposons replicate by a mechanism which resembles that of the retroviruses. Boeke and Sandmeyer, (1991) Yeast transposable elements. In The Molecular and Cellular Biology of the Yeast Saccharomyces, edited by J. Broach, E. Jones and J. Pringle, pp. 193-261. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. They typically use a specific tRNA to prime reverse transcription, and a linear cDNA is synthesized through a series of template transfers that require redundant LTR sequences at each end of the element mRNA. This all occurs within a virus-like particle formed from proteins encoded by the retrotransposon mRNA. After reverse transcription, an integration complex is organized that directs the resulting cDNA to a new site in the genome of the host cell.
Phylogenetic analyses based on reverse transcriptase amino acid sequences resolve the LTR retrotransposons into two families: the Ty3/gypsy retrotransposons (Metaviridae), and the Ty1/copia elements (Pseudoviridae). Boeke et al., (1998) Metaviridae. In Virus Taxonomy: ICTV VIIth Report, edited by F. A. Murphy. Springer-Verlag, N.Y.; Boeke et al. (1998) Pseudoviridae. In Virus Taxonomy: ICTV VIIth Report, edited by F. A. Murphy. Springer Verlag, N.Y.; Xiong and Eickbush (1990) EMBO J. 9: 3353-3362. Although distinct, Ty3/gypsy elements are more closely related to the retroviruses than to the Ty1/copia elements. They also share a similar genetic organization with the retroviruses, principally in the order of integrase and reverse transcriptase in their pol genes. For the Ty3/gypsy elements, reverse transcriptase precedes integrase, and this order is reversed for the Ty1/copia elements. In addition, some Ty3/gypsy elements have an extra open reading frame (ORF) similar to retroviral envelope (env) proteins, which is required for viral infectivity. The Drosophila melanogaster gypsy retrotransposons encode an env-like ORF and can be transmitted between cells. Kim et al. (1994) Proc. Natl. Acad. Sci. USA 91: 1285-1289; Song et al. (1994) Genes and Dev. 8: 2046-2057. Thus there are two distinct lineages of infectious LTR retroelements, the retroviruses and those Ty3/gypsy retrotransposons that encode envelope-like proteins. The Ty3/gypsy elements have been divided into two genera, the metaviruses and the errantiviruses, the latter of which include all elements with env-like genes. Boeke et al., (1998) Metaviridae. In Virus Taxonomy: ICTV VlIth Report, edited by F. A. Murphy. Springer-Verlag, N.Y.
In plants, retrotransposons have been extremely successful. Bennetzen (1996) Trends Microbiol. 4: 347-353; Voytas (1996) Genetics 142: 569-578. The enormous size of many plant genomes demonstrates a great tolerance for repetitive DNA, a substantial proportion of which appears to be composed of retrotransposons. Because of their abundance, retrotransposons have undoubtedly influenced plant gene evolution. They can cause mutations in coding sequences (Grandbastien et al. (1989) Nature 337: 376-380; Hirochika et al. (1996) Proc. Natl. Acad. Sci. USA 93: 7783-7788; Purugganan and Wessler (1994) Proc. Natl. Acad. Sci. USA 91: 11674-11678), and the promoter regions of some plant genes contain relics of retrotransposon insertions that contribute transcriptional regulatory sequences. White et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11792-11796. Retrotransposons also generate gene duplications: Repetitive retrotransposon sequences provide substrates for unequal crossing over, and such an event is thought to have caused a zein gene duplication in maize. White et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11792-11796. Occasionally, cellular mRNAs are reverse transcribed and the resultant cDNA recombines into the genome giving rise to new genes, or more frequently, cDNA pseudogenes. Maestre et al. (1995) EMBO J. 14: 6333-6338. The transduction of gene sequences during reverse transcription, which produced the oncogenic retroviruses, has also been documented to occur for a plant retrotransposon (Bureau et al. (1994) Cell 77: 479-480; Jin and Bennetzen (1994) Plant Cell 6: 1177 1186); a maize Bsl insertion in Adhl carries part of an ATPase gene and is the only known example of a retrotransposon-mediated gene transduction event.
Plant genomes encode representatives of the two major lineages of LTR retrotransposons that have been identified in other eukaryotes. Among these are numerous examples of Ty 1/copia elements (e.g. Konieczny et al. (1991) Genetics 127: 801-809; Voytas and Ausubel (1988) Nature 336: 242-244; Voytas et al. (1990) Genetics 126: 713-721) Also prevalent are Ty3/gypsy elements, which are members of the genus Metaviridae (Smyth et al. 1989; Purugganan and Wessler 1994 Proc. Natl. Acad. Sci. USA 91: 11674-11678; Su and Brown 1997). As stated above, the metaviruses do not encode an envelope protein characteristic of the retroviruses. It has been suggested that some plant retrovirus-like elements may have lost, or not yet gained, genes such as the envelope gene required for cell-to-cell transmission (Bennetzen (1996) Trends Microbiol. 4: 347-353). As one group of researchers described the uncertainty, xe2x80x9c[s]ince genes encoding ENV [envelope] functions are very heterogeneous at the sequence level and difficult to identify by homology even between retroviruses, the possibility cannot be completely excluded at the present time that the 3xe2x80x2 ORF of Cyclops [the retrotransposon described in the paper] is, in fact, an env gene and, hence, Cyclops is a retrovirus or a descendant of one.xe2x80x9d Chavanne et al. (1998) Plant Molecular Biol 37: 363-375.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on subjective characterization of information available to the applicant, and does not constitute any admission as to the accuracy of the dates or contents of these documents.
In general, the present invention provides materials, such as nucleic acids, vectors, cells, and plants (including plant parts, seeds, embryos, etc.), and methods to manipulate the materials. In particular, molecular tools are provided in the form of retroelements and retroelement-containing vectors, cells and plants. The particular methods include methods to introduce the retroelements into cells, especially wherein the retroelements carries at least one agronomically-significant characteristic. The best mode of the present invention is a particular method to transfer agronomically-significant characteristics to plants wherein a helper cell line which expresses gag, pol and env sequences is used to enable transfer of a secondary construct which carries an agronomically-significant characteristic and has retroelement sequences that allow for replication and integration.
In one embodiment, there are provided isolated nucleic acid molecules, wherein said nucleic acid molecules encode at least a portion of a plant retroelement and comprises a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence which is a plant retroelement primer binding site and which has more than 95% identity to SEQ ID NO 2, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which is at least a portion of a plant retroelement envelope sequence and which has more than 50% identity to SEQ ID NO 5, wherein said identity can be determined using the DNAsis computer program and default parameters;
(c) a nucleic acid sequence which is at least a portion of a plant retroelement gag sequence and which has more than 50% identity to SEQ ID NO 7, wherein said identity can be determined using the DNAsis computer program and default parameters;
(d) a nucleic acid sequence which is at least a portion of a plant retroelement integrase sequence and which has more than 70% identity to SEQ ID NO 9, wherein said identity can be determined using the DNAsis computer program and default parameters;
(e) a nucleic acid sequence which is at least a portion of a plant retroelement reverse transcriptase sequence and which has more than 70% identity to SEQ ID NO 11, wherein said identity can be determined using the DNAsis computer program and default parameters;
(f) a nucleic acid sequence which is at least a portion of a plant retroelement protease sequence and which has more than 50% identity to SEQ ID NO 13, wherein said identity can be determined using the DNAsis computer program and default parameters;
(g) a nucleic acid sequence which is at least a portion of a plant retroelement RNAseH sequence and which has more than 70% identity to SEQ ID NO 15, wherein said identity can be determined using the DNAsis computer program and default parameters;
(h) a nucleic acid sequence which is at least a portion of a plant retroelement sequence and which has more than 50% identity to SEQ ID NO 17, wherein said identity can be determined using the DNAsis computer program and default parameters;
(i) a nucleic acid sequence which is selected from the group consisting of: SEQ ID NO 2; SEQ ID NO 5; SEQ ID NO 7; SEQ ID NO 9; SEQ ID NO 11; SEQ ID NO 13; SEQ ID NO 15; and SEQ ID NO 17.
(j) a nucleic acid sequence which encodes an amino acid sequence which is at least a portion of a plant retroelement envelope sequence and has more than 30% identity to SEQ ID NO 6, wherein said identity can be determined using the DNAsis computer program and default parameters;
(k) a nucleic acid sequence which encodes an amino acid sequence which is at least a portion of a plant retroelement gag sequence and has more than 30% identity to SEQ ID NO 8, wherein said identity can be determined using the DNAsis computer program and default parameters;
(l) a nucleic acid sequence which encodes an amino acid sequence which is at least a portion of a plant retroelement integrase sequence and has more than 75% identity to SEQ ID NO 10, wherein said identity can be determined using the DNAsis computer program and default parameters;
(m) a nucleic acid sequence which encodes an amino acid sequence which is at least a portion of a plant retroelement reverse transcriptase sequence and has more than 79% identity to SEQ ID NO 12, wherein said identity can be determined using the DNAsis computer program and default parameters;
(n) a nucleic acid sequence which encodes an amino acid sequence which is at least a portion of a plant retroelement protease sequence and has more than 55% identity to SEQ ID NO 14, wherein said identity can be determined using the DNAsis computer program and default parameters;
(o) a nucleic acid sequence which encodes an amino acid sequence which is at least a portion of a plant retroelement RNAseH sequence and has more than 90% identity to SEQ ID NO 16, wherein said identity can be determined using the DNAsis computer program and default parameters;
(p) a nucleic acid sequence which encodes an amino acid sequence which is at least a portion of a plant retroelement sequence and has more than 40% identity to SEQ ID NO 18, wherein said identity can be determined using the DNAsis computer program;
(q) a nucleic acid sequence which encodes an amino acid sequence selected from the group consisting of: SEQ ID NO 4; SEQ ID NO 6; SEQ ID NO 8; SEQ ID NO 10; SEQ ID NO 12; SEQ ID NO 14; SEQ ID NO 16; and SEQ ID NO 18;
(r) a nucleic acid sequence which encodes an allelic variant of an amino acid sequence selected from the group consisting of: SEQ ID NO 4; SEQ ID NO 6; SEQ ID NO 8; SEQ ID NO 10; SEQ ID NO 12; SEQ ID NO 14; SEQ ID NO 16; and SEQ ID NO 18; and
(s) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); a nucleic acid sequence of (c); a nucleic acid sequence of (d); a nucleic acid sequence of (e); a nucleic acid sequence of (f); a nucleic acid sequence of (g); a nucleic acid sequence of (h); a nucleic acid sequence of (i); a nucleic acid sequence of (j); a nucleic acid sequence of (k); a nucleic acid sequence of (l); a nucleic acid sequence of (m); a nucleic acid sequence of (n); a nucleic acid sequence of (o); a nucleic acid sequence of (p); a nucleic acid sequence of (q); and a nucleic acid sequence of (r).
Seeds and plants comprising a nucleic acid as above are particularly provided. Nucleic acid molecules as above which comprise gag, pol and env genes and which comprise adenine-thymidine-guanidine as the gag gene start codon are also particularly provided. Those which comprise gag, pol and env genes, the adenine-thymidine-guanidine as the gag gene start codon, and which further comprises SEQ ID NO 4 are also provided.
Plant envelope sequences and constructs which comprise the sequences are provided, as are cells, seeds, embryos and plants comprising them. Preferred are isolated nucleic acid molecules, wherein said nucleic acid molecules encode at least a portion of a plant envelope sequence and comprises a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence which has more than 90% identity to SEQ ID NO 5, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which encodes SEQ ID NO 5;
(c) a nucleic acid sequence which encodes an amino acid sequence which has greater than 85% identity to SEQ ID NO 6, wherein said identity can be determined using the DNAsis computer program and default parameters;
(d) a nucleic acid sequence which encodes amino acid sequence SEQ ID NO 6;
(e) a nucleic acid sequence which encodes an allelic variant of SEQ ID NO 6; and
(f) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); a nucleic acid sequence of (c); a nucleic acid sequence of (d); and a nucleic acid sequence of (e).
Plant cells comprising an isolated nucleic acid molecule above are particularly preferred. Also preferred are plant envelope proteins comprising an amino acid sequence encoded by the above. Methods to impart agronomically-significant characteristics to at least one plant cell are also provided, comprising: contacting a plant envelope protein as described to at least one plant cell under conditions sufficient to allow a nucleic acid molecule to enter said cell, wherein said nucleic acid molecule encodes an agronomically-significant characteristic.
Plant integrase sequences and constructs which comprise the sequences are provided, as are cells, seeds, embryos and plants comprising them. Preferred are isolated nucleic acid molecules, wherein said nucleic acid molecules encode at least a portion of a plant integrase sequence and comprises a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence which has more than 90% identity to SEQ ID NO 9, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which encodes SEQ ID NO 9;
(c) a nucleic acid sequence which encodes an amino acid sequence which has greater than 85% identity to SEQ ID NO 10, wherein said identity can be determined using the DNAsis computer program and default parameters;
(d) a nucleic acid sequence which encodes amino acid sequence SEQ ID NO 10;
(e) a nucleic acid sequence which encodes an allelic variant of SEQ ID NO 10; and
(f) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); a nucleic acid sequence of (c); a nucleic acid sequence of (d); and a nucleic acid sequence of (e).
Plant cells comprising an isolated nucleic acid molecule above are particularly preferred. Also preferred are plant integrase proteins comprising an amino acid sequence encoded by the above. Methods to impart agronomically-significant characteristics to at least one plant cell are also provided, comprising: contacting a plant integrase protein as described to at least one plant cell under conditions sufficient to allow a nucleic acid molecule to enter said cell, wherein said nucleic acid molecule encodes an agronomically-significant characteristic.
Plant reverse transcriptase sequences and constructs which comprise the sequences are provided, as are cells, seeds, embryos and plants comprising them. Preferred are isolated nucleic acid molecules, wherein said nucleic acid molecules encode at least a portion of a plant reverse transcriptase sequence and comprises a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence which has more than 90% identity to SEQ ID NO 11, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which encodes SEQ ID NO 11;
(c) a nucleic acid sequence which encodes an amino acid sequence which has greater than 85% identity to SEQ ID NO 12, wherein said identity can be determined using the DNAsis computer program and default parameters;
(d) a nucleic acid sequence which encodes amino acid sequence SEQ ID NO 12;
(e) a nucleic acid sequence which encodes an allelic variant of SEQ ID NO 12; and
(f) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); a nucleic acid sequence of (c); a nucleic acid sequence of (d); and a nucleic acid sequence of (e).
Plant cells comprising an isolated nucleic acid molecule above are particularly preferred. Also preferred are plant reverse transcriptase proteins comprising an amino acid sequence encoded by the above. Methods to impart agronomically-significant characteristics to at least one plant cell are also provided, comprising; contacting a plant reverse transcriptase protein as described to at least one plant cell under conditions sufficient to allow a nucleic acid molecule to enter said cell, wherein said nucleic acid molecule encodes an agronomically-significant characteristic.
Plant RNAseH sequences and constructs which comprise the sequences are provided, as are cells, seeds, embryos and plants comprising them. Preferred are isolated nucleic acid molecules, wherein said nucleic acid molecules encode at least a portion of a plant RNAseH sequence and comprises a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence which has more than 90% identity to SEQ ID NO 15, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which encodes SEQ ID NO 15;
(c) a nucleic acid sequence which encodes an amino acid sequence which has greater than 95% identity to SEQ ID NO 16, wherein said identity can be determined using the DNAsis computer program and default parameters;
(d) a nucleic acid sequence which encodes amino acid sequence SEQ ID NO 16;
(e) a nucleic acid sequence which encodes an allelic variant of SEQ ID NO 16; and
(f) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); a nucleic acid sequence of (c); a nucleic acid sequence of (d); and a nucleic acid sequence of (e).
Plant cells comprising an isolated nucleic acid molecule above are particularly preferred. Also preferred are plant RNAseH proteins comprising an amino acid sequence encoded by the above. Methods to impart agronomically-significant characteristics to at least one plant cell are also provided, comprising: contacting a plant RNAseH protein as described to at least one plant cell under conditions sufficient to allow a nucleic acid molecule to enter said cell, wherein said nucleic acid molecule encodes an agronomically-significant characteristic.
Plant retroelement sequences and constructs which comprise the sequences are provided, as are cells, seeds, embryos and plants comprising them. Preferred are isolated nucleic acid molecules, wherein said nucleic acid molecules encode at least a portion of a plant retroelement sequence and comprises a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence which has more than 95% identity to a nucleic acid sequence selected from the group consisting of: SEQ ID NO 2; SEQ ID NO 5; SEQ ID NO 7; SEQ ID NO 9; SEQ ID NO 11; SEQ ID NO 13; SEQ ID NO 15; and SEQ ID NO 17, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which is selected from the group consisting of: SEQ ID NO 2; SEQ ID NO 5; SEQ ID NO 7; SEQ ID NO 9; SEQ ID NO 11; SEQ ID NO 13; SEQ ID NO 15; and SEQ ID NO 17;
(c) a nucleic acid sequence which encodes an amino acid sequence which has more than 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NO 4; SEQ ID NO 6; SEQ ID NO 8; SEQ ID NO 10; SEQ ID NO 12; SEQ ID NO 14; SEQ ID NO 16; SEQ ID NO 18, wherein said identity can be determined using the DNAsis computer program and default parameters;
(d) a nucleic acid sequence which encodes an amino acid sequence selected from the group consisting of: SEQ ID NO 4; SEQ ID NO 6; SEQ ID NO 8; SEQ ID NO 10; SEQ ID NO 12; SEQ ID NO 14; SEQ ID NO 16; and SEQ ID NO 18;
(e) a nucleic acid sequence which encodes an allelic variant of an amino acid sequence selected from the group consisting of: SEQ ID NO 4; SEQ ID NO 6; SEQ ID NO 8; SEQ ID NO 10; SEQ ID NO 12; SEQ ID NO 14; SEQ ID NO 16; and SEQ ID NO 18; and
(f) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); a nucleic acid sequence of (c); a nucleic acid sequence of (d); and a nucleic acid sequence of (e).
Nucleic acid molecule as above, which further comprises at least one nucleic acid sequence which encodes at least one agronomically-significant characteristic are preferred. More preferred are those nucleic acid molecules as described wherein the agronomically-significant characteristic is selected from the group consisting of: male sterility; self-incompatibility; foreign organism resistance; improved biosynthetic pathways; environmental tolerance; photosynthetic pathways; and nutrient content and those wherein the agronomically significant characteristic is selected from the group consisting of: fruit ripening; oil biosynthesis; pigment biosynthesis; seed formation; starch metabolism; salt tolerance; cold/frost tolerance; drought tolerance; tolerance to anaerobic conditions; protein content; carbohydrate content (including sugars and starches); amino acid content; and fatty acid content.
Seeds and plants comprising a nucleic acid molecule as described are also preferred. More preferred are plants as described, wherein the plant is selected from the group consisting of: soybean; maize; sugar cane; beet; tobacco; wheat; barley; poppy; rape; sunflower; alfalfa; sorghum; rose; carnation; gerbera; carrot; tomato; lettuce; chicory; pepper; melon; cabbage; oat; rye; cotton; flax; potato; pine; walnut; citrus (including oranges, grapefruit etc.); hemp; oak; rice; petunia; orchids; Arabidopsis; broccoli; cauliflower, brussel sprouts; onion; garlic; leek; squash; pumpkin; celery; pea; bean (including various legumes); strawberries; grapes; apples; pears; peaches; banana; palm; cocoa; cucumber, pineapple; apricot; plum; sugar beet; lawn grasses; maple; triticale; safflower, peanut; and olive. Most preferred are plants as described which are soybean plants.
Plant retroelements comprising an amino acid sequence encoded by a nucleic acid sequence described are also provided. Plant cells comprising a nucleic acid molecule described herein, as well as plant retroviral proteins encoded by nucleic acid molecules described herein are provided.
Moreover, methods to transfer nucleic acid into a plant cell, comprising contacting a nucleic acid molecule of the present invention with at least one plant cell under conditions sufficient to allow said nucleic acid molecule to enter at least one cell of said plant are provided. In particular there is provided, methods to impart agronomically-significant characteristics to at least one plant cell, comprising: contacting a plant retroelement of the present invention to at least one plant cell under conditions sufficient to allow a nucleic acid molecule to enter said cell, wherein said nucleic acid molecule encodes an agronomically-significant characteristic. Methods as described, wherein the agronomically-significant characteristic is selected from the group consisting of: male sterility; self-incompatibility; foreign organism resistance; improved biosynthetic pathways; environmental tolerance; photosynthetic pathways; and nutrient content and those wherein the agronomically significant characteristic is selected from the group consisting of: fruit ripening; oil biosynthesis; pigment biosynthesis; seed formation; starch metabolism; salt tolerance; cold/frost tolerance; drought tolerance; tolerance to anaerobic conditions; protein content; carbohydrate content (including sugars and starches); amino acid content; and fatty acid content.
Plant retroelement sequences comprising specialized signals, and constructs which comprise the sequences are provided, as are cells, seeds, embryos and plants comprising them. Preferred are isolated nucleic acid molecules, comprising a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence which has more than 95% identity to SEQ ID NO 2; wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which is SEQ ID NO 2;
(c) a nucleic acid sequence which encodes amino acid sequence SEQ ID NO 4; and
(d) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); and a nucleic acid sequence of (c).
Plant retroelements as described above, which further comprise at least one nucleic acid sequence which encodes at least one agronomically-significant characteristic are preferred. More preferred are those methods wherein the agronomically-significant characteristic is selected from the group consisting of: male sterility; self-incompatibility; foreign organism resistance; improved biosynthetic pathways; environmental tolerance; photosynthetic pathways; and nutrient content and those wherein the agronomically significant characteristic is selected from the group consisting of: fruit ripening; oil biosynthesis; pigment biosynthesis; seed formation; starch metabolism; salt tolerance; cold/frost tolerance; drought tolerance; tolerance to anaerobic conditions; protein content; carbohydrate content (including sugars and starches); amino acid content; and fatty acid content.
Preferred are plant retroviral particles comprising an isolated retroelement as described, and seeds and plants comprising the retroelements as described. More preferred plants include soybean; maize; sugar cane; beet; tobacco; wheat; barley; poppy; rape; sunflower; alfalfa; sorghum; rose; carnation; gerbera; carrot; tomato; lettuce; chicory; pepper, melon; cabbage; oat; rye; cotton; flax; potato; pine; walnut; citrus (including oranges, grapefruit etc.); hemp; oak; rice; petunia; orchids; Arabidopsis; broccoli; cauliflower, brussel sprouts; onion; garlic; leek; squash; pumpkin; celery; pea; bean (including various legumes); strawberries; grapes; apples; pears; peaches; banana; palm; cocoa; cucumber, pineapple; apricot; plum; sugar beet; lawn grasses; maple; triticale; safflower; peanut; and olive. Soybean is most preferred.
Also provided are methods to transfer nucleic acid into a plant cell, comprising contacting a plant retroelement as described with at least one plant cell under conditions sufficient to allow said plant retroelement to enter said cell. Methods to impart agronomically-significant characteristics to a plant, comprising contacting a plant retroelement as described with at least one plant cell under conditions sufficient to allow said plant retroelement to enter said cell are also preferred. Those methods wherein the plant retroelement is contacted with said cell via a plant retroviral particle described herein are preferred.
Plant retroviruses are also provided. In particular, plant retroviral particles comprising a plant-derived retrovirus envelope protein are provided. Plant retroviral particles comprising a plant-derived retrovirus envelope protein and which further comprise a plant retroviral protein selected from the group consisting of: plant-derived integrase; plant derived reverse transcriptase; plant-derived gag; and plant-derived RNAseH are preferred.
Plant retroviral particles comprising specialized retroviral proteins, and cells, seeds, embryos and plants which comprise the retroviral particles are provided. Preferred are isolated retroviral particles comprising a plant retroviral protein encoded by a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence comprising (i) a nucleic acid sequence which encodes at least one plant retroviral envelope protein, and (ii) a nucleic acid sequence which has more than 60% identity to a nucleic acid sequence selected from the group consisting of: SEQ ID NO 9; SEQ ID NO 11; SEQ ID NO 15; SEQ ID NO 26; SEQ ID NO 27; SEQ ID NO 28; SEQ ID NO 29; SEQ D NO 30; and SEQ ID NO 31, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which encodes an amino acid sequence encoded by a nucleic acid sequence (a);
(c) a nucleic acid sequence which encodes an allelic variant of an amino acid sequence encoded by a nucleic acid sequence of (a); and
(d) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); and a nucleic acid sequence of (c).
In particular, there are provided plant retroviral particles, wherein said nucleic acid sequence as described in (a) comprises a plant envelope nucleic acid specifically mentioned in claim 6 is preferred. Those particles which further comprise at least one nucleic acid sequence which encodes at least one agronomically-significant characteristic are preferred.
Also provided are methods to transfer nucleic acid into a plant cell, comprising contacting a plant retroviral particle as described above to at least one plant cell under conditions sufficient to allow said nucleic acid to enter said cell. More preferred are methods to impart agronomically-significant characteristics to a plant, comprising contacting a plant retroviral particle as described to at least one plant cell under conditions sufficient to allow said nucleic acid to enter said cell.
More preferred are isolated retroviral particles comprising a plant retroviral protein encoded by a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence which has more than 80% identity to a nucleic acid sequence selected from the group consisting of: SEQ ID NO 9; SEQ ID NO 11; and SEQ ID NO 15, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which encodes a nucleic acid selected from the group consisting of: SEQ ID NO 9; SEQ ID NO 11; and SEQ ID NO 15;
(c) a nucleic acid sequence which encodes an amino acid sequence encoded by a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); and a nucleic acid sequence of (b);
(d) a nucleic acid sequence which encodes an allelic variant of an amino acid sequence encoded by a nucleic acid selected from the group consisting of: a nucleic acid sequence of (a); and a nucleic acid sequence of (b); and
(e) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); a nucleic acid sequence of (c); and a nucleic acid sequence of (d).
Nucleic acids as above, which further comprises at least one nucleic acid sequence which encodes at least one agronomically-significant characteristic are preferred. More preferred are those nucleic acids wherein the agronomically-significant characteristic is selected from the group consisting of: male sterility; self-incompatibility; foreign organism resistance; improved biosynthetic pathways; environmental tolerance; photosynthetic pathways; and nutrient content. Also more preferred are those isolated nucleic acid molecule as described, wherein the agronomically significant characteristic is selected from the group consisting of: fruit ripening; oil biosynthesis; pigment biosynthesis; seed formation; starch metabolism; salt tolerance; cold/frost tolerance; drought tolerance; tolerance to anaerobic conditions; protein content; carbohydrate content (including sugars and starches); amino acid content; and fatty acid content.
Also provided are methods to transfer nucleic acid into a plant cell, comprising contacting a plant retroviral particle as described above to at least one plant cell under conditions sufficient to allow said nucleic acid to enter said cell. More preferred are methods to impart agronomically-significant characteristics to a plant, comprising contacting a plant retroviral particle as described to at least one plant cell under conditions sufficient to allow said nucleic acid to enter said cell.
Also preferred are isolated retroviral particles comprising a plant retroviral protein encoded by a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence which has more than 60% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO 9; SEQ ID NO 11; SEQ ID NO 15; SEQ ID NO 26; SEQ ID NO 27; SEQ ID NO 28; SEQ ID NO 29; SEQ ID NO 30; and SEQ ID NO 31, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which encodes a nucleic acid selected from the group consisting of: SEQ ID NO 9; SEQ ID NO 11; SEQ ID NO 15; SEQ ID NO 26; SEQ ID NO 27; SEQ ID NO28; SEQ ID NO 29; SEQ ID NO 30; and SEQ ID NO 31;
(c) a nucleic acid sequence which encodes an amino acid sequence encoded by a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); and a nucleic acid sequence of (b);
(d) a nucleic acid sequence which encodes an allelic variant of an amino acid sequence encoded by a nucleic acid selected from the group consisting of: a nucleic acid sequence of (a); and a nucleic acid sequence of (b); and
(e) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); a nucleic acid sequence of (c); and a nucleic acid sequence of (d).
Plant retroviral particles as described above, which further comprises an envelope-encoding nucleic acid sequence specifically described herein are preferred. Preferred are those retroviral particles which further comprise at least one nucleic acid sequence which encodes at least one agronomically-significant characteristic.
Also provided are methods to transfer nucleic acid into a plant cell, comprising contacting a plant retroviral particle as described above to at least one plant cell under conditions sufficient to allow said nucleic acid to enter said cell. More preferred are methods to impart agronomically-significant characteristics to a plant, comprising contacting a plant retroviral particle as described to at least one plant cell under conditions sufficient to allow said nucleic acid to enter said cell.
Also provided by the present invention are isolated nucleic acid molecules, wherein said nucleic acid molecule encodes at least a portion of a plant retroelement reverse transcriptase and comprises a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence having more than 85% identity to a nucleic acid sequence selected from the group consisting of even-numbered SEQ ID NOs inclusive from SEQ ID NO 42 to SEQ ID NO 164, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which encodes an amino acid sequence having more than 85% identity to an amino acid sequence selected from the group consisting of odd-numbered SEQ ID NOs inclusive from SEQ ID NO 43 through SEQ ID NO 165, wherein said identity can be determined using the DNAsis computer program and default parameters;
(c) a nucleic acid sequence which encodes an allelic variant of a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b).
(d) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b).
Seeds and plants comprising the nucleic acid molecules are also provided, as are nucleic acids as described which comprise gag, pol and env genes and which comprises adenine-thymidine-guanidine as the gag gene start codon. Moreover, those nucleic acids which further comprises SEQ ID NO 5 are also provided. Also provided by the present invention are isolated nucleic acid molecules described, wherein said nucleic acid molecule encodes at least a portion of a plant envelope sequence and comprises a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence which has more than 90% identity to SEQ ID NO 5, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which encodes an amino acid sequence which has greater than 85% identity to SEQ ID NO 6, wherein said identity can be determined using the DNAsis computer program and default pararmeters;
(c) a nucleic acid sequence which encodes an allelic variant of SEQ ID NO 5; and
(d) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); and a nucleic acid sequence of c).
Plant cells comprising this embodiment are also provided. Methods to impart agronomically-significant characteristics to at least one plant cell, comprising:
contacting a nucleic acid molecule described to at least one plant cell under conditions sufficient to allow at least one agronomically-significant nucleic acid molecule to enter said cell.
Also part of the present invention are isolated nucleic acid molecules, wherein said nucleic acid molecule encodes at least a portion of a plant retroelement reverse transcriptase and comprises a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence having more than 95% identity to a nucleic acid sequence selected from the group consisting of even-numbered SEQ ID NOs inclusive from SEQ ID NO 42 to SEQ ID NO 164, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which encodes an amino acid sequence having more than 95% identity to an amino acid sequence selected from the group consisting of odd-numbered SEQ ID NOs inclusive from SEQ ID NO 43 through SEQ ID NO 165, wherein said identity can be determined using the DNAsis computer program and default parameters;
(c) a nucleic acid sequence which encodes an allelic variant of a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b).
(d) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b).
Seeds and plants comprising the nucleic acid molecules are also provided, as are nucleic acids as described which comprise gag, pol and env genes and which comprises adenine-thymidine-guanidine as the gag gene start codon. Moreover, those nucleic acids which further comprises SEQ ID NO 5 are also provided. Methods to impart agronomically-significant characteristics to at least one plant cell, comprising:
contacting a nucleic acid molecule described to at least one plant cell under conditions sufficient to allow at least one agronomically-significant nucleic acid molecule to enter said cell.
Also provided are isolated nucleic acid molecule, wherein said nucleic acid molecule encodes at least a portion of a plant retroelement reverse transcriptase and comprises a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence selected from the group consisting of even-numbered SEQ ID NOs inclusive from SEQ ID NO 42 to SEQ ID NO 164, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which encodes an amino acid sequence selected from the group consisting of odd-numbered SEQ ID NOs inclusive from SEQ ID NO 43 through SEQ ID NO 165, wherein said identity can be determined using the DNAsis computer program and default parameters;
(c) a nucleic acid sequence which encodes an allelic variant of a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b).
(d) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b).
Seeds and plants comprising the nucleic acid molecules are also provided, as are nucleic acids as described which comprise gag, pol and env genes and which comprises adenine-thymidine-guanidine as the gag gene start codon. Moreover, those nucleic acids which further comprises SEQ ID NO 5 are also provided. Methods to impart agronomically-significant characteristics to at least one plant cell, comprising:
contacting a nucleic acid molecule described to at least one plant cell under conditions sufficient to allow at least one agronomically-significant nucleic acid molecule to enter said cell.
Nucleic acid molecules of the present invention which further comprise at least one nucleic acid sequence which encodes at least one agronomically-significant characteristic are also provided. Those nucleic acid molecules wherein the agronomically-significant characteristic is selected from the group consisting of: male sterility; self-incompatibility; foreign organism resistance; improved biosynthetic pathways; environmental tolerance; photosynthetic pathways; and nutrient content are preferred. Also preferred are those nucleic acid molecules wherein the agronomically significant characteristic is selected from the group consisting of: fruit ripening; oil biosynthesis; pigment biosynthesis; seed formation; starch metabolism; salt tolerance; cold/frost tolerance; drought tolerance; tolerance to anaerobic conditions; protein content; carbohydrate content (including sugars and starches); amino acid content; and fatty acid content.
Also provided are isolated plant retroviral particles comprising a nucleic acid molecule of the present invention.
Preferred plants are selected from the group consisting of: soybean; maize; sugar cane; beet; tobacco; wheat; barley; poppy; rape; sunflower, alfalfa; sorghum; rose; carnation; gerbera; carrot; tomato; lettuce; chicory; pepper, melon; cabbage; oat; rye; cotton; flax; potato; pine; walnut; citrus (including oranges, grapefruit etc.); hemp; oak; rice; petunia; orchids; Arabidopsis; broccoli; cauliflower; brussel sprouts; onion; garlic; leek; squash; pumpkin; celery; pea; bean (including various legumes); strawberries; grapes; apples; pears; peaches; banana; palm; cocoa; cucumber, pineapple; apricot; plum; sugar beet; lawn grasses; maple; triticale; safflower, peanut; and olive.
In the present invention, it is preferred that the nucleic acid sequences are transmissible to either all plants, or to a limited set of plants, such as a species. For instance, plant viruses in general only infect a narrow host range or maybe infect a single species, and the present compounds may be genetically engineered to be similar. However, if a broad host range is desirable, those features which cause specificity can be removed or overridden by the feature of broad transmissibility. The present invention is drawn to both these embodiments, as well as other variations.
xe2x80x9cAllelic variantxe2x80x9d is meant to refer to a full length gene or partial sequence of a full length gene that occurs at essentially the same locus (or loci) as the referent sequence, but which, due to natural variations caused by, for example, mutation or recombination, has a similar but not identical sequence. Allelic variants typically encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared. Allelic variants can also comprise alterations in the 5xe2x80x2 or 3xe2x80x2 untranslated regions of the gene (e.g., in regulatory control regions).
By xe2x80x9cagronomically-significantxe2x80x9d it is meant any trait of a plant which is recognized by members of the agricultural industry as desirable.
xe2x80x9cFragmentxe2x80x9d is meant to refer to any subset of the referent nucleic acid molecule.
By xe2x80x9cplantxe2x80x9d it is meant one or more plant seed, plant embryo, plant part or whole plant. The plant may be an angiosperm (monocot or dicot), gymnosperm, man-made or naturally-occurring.
By xe2x80x9cproteinsxe2x80x9d it is meant any compounds which comprise amino acids, including peptides, polypeptides, fusion proteins, etc.
Moreover, for the purposes of the present invention, the term xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d entity refers to one or more of that entity; for example, xe2x80x9ca proteinxe2x80x9d or xe2x80x9ca nucleic acid moleculexe2x80x9d refers to one or more of those compounds or at least one compound. As such, the terms xe2x80x9caxe2x80x9d (or xe2x80x9canxe2x80x9d), xe2x80x9cone or morexe2x80x9d and xe2x80x9cat least onexe2x80x9d can be used interchangeably herein. It is also to be noted that the terms xe2x80x9ccomprisingxe2x80x9d, xe2x80x9cincludingxe2x80x9d, and xe2x80x9chavingxe2x80x9d can be used interchangeably. Furthermore, a compound xe2x80x9cselected from the group consisting ofxe2x80x9d refers to one or more of the compounds in the list that follows, including mixtures (i.e., combinations) of two or more of the compounds. According to the present invention, an isolated, or biologically pure, protein or nucleic acid molecule is a compound that has been removed from its natural milieu. As such, xe2x80x9cisolatedxe2x80x9d and xe2x80x9cbiologically purexe2x80x9d do not necessarily reflect the extent to which the compound has been purified. An isolated compound of the present invention can be obtained from its natural source, can be produced using molecular biology techniques or can be produced by chemical synthesis. Lastly, xe2x80x9cmore thanxe2x80x9d and xe2x80x9cgreater thanxe2x80x9d are interchangeable, and when used to modify a percent identity, ie. xe2x80x9cmore than 90% identityxe2x80x9d, mean any increment to 100%, so long as the increment were greater than the percentage specifically named. In the example of xe2x80x9cmore than 90% identityxe2x80x9d, the term would include, among all other possibilities, 90.00001, 93.7, 98.04 and 99. 0827 and 100%.
The following is a summary of the sequence listing, as a convenient reference.
In one embodiment, there are provided isolated nucleic acid molecules, wherein said nucleic acid molecules encode at least a portion of a plant retroelement and comprises a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence which is a plant retroelement primer binding site and which has more than 95% identity to SEQ ID NO 2, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which is at least a portion of a plant retroelement envelope sequence and which has more than 50% identity to SEQ ID NO 5, wherein said identity can be determined using the DNAsis computer program and default parameters;
(c) a nucleic acid sequence which is at least a portion of a plant retroelement gag sequence and which has more than 50% identity to SEQ ID NO 7, wherein said identity can be determined using the DNAsis computer program and default parameters;
(d) a nucleic acid sequence which is at least a portion of a plant retroelement integrase sequence and which has more than 70% identity to SEQ ID NO 9, wherein said identity can be determined using the DNAsis computer program and default parameters;
(e) a nucleic acid sequence which is at least a portion of a plant retroelement reverse transcriptase sequence and which has more than 70% identity to SEQ ID NO 11, wherein said identity can be determined using the DNAsis computer program and default parameters;
(f) a nucleic acid sequence which is at least a portion of a plant retroelement protease sequence and which has more than 50% identity to SEQ ID NO 13, wherein said identity can be determined using the DNAsis computer program and default parameters;
(g) a nucleic acid sequence which is at least a portion of a plant retroelement RNAseH sequence and which has more than 70% identity to SEQ ID NO 15, wherein said identity can be determined using the DNAsis computer program and default parameters;
(h) a nucleic acid sequence which is at least a portion of a plant retroelement sequence and which has more than 50% identity to SEQ ID NO 17, wherein said identity can be determined using the DNAsis computer program and default parameters;
(i) a nucleic acid sequence which is selected from the group consisting of: SEQ ID NO 2; SEQ ID NO 5; SEQ ID NO 7; SEQ ID NO 9; SEQ ID NO 11; SEQ ID NO 13; SEQ ID NO 15; and SEQ ID NO 17.
(j) a nucleic acid sequence which encodes an amino acid sequence which is at least a portion of a plant retroelement envelope sequence and has more than 30% identity to SEQ ID NO 6, wherein said identity can be determined using the DNAsis computer program and default parameters;
(k) a nucleic acid sequence which encodes an amino acid sequence which is at least a portion of a plant retroelement gag sequence and has more than 30% identity to SEQ ID NO 8, wherein said identity can be determined using the DNAsis computer program and default parameters;
(l) a nucleic acid sequence which encodes an amino acid sequence which is at least a portion of a plant retroelement integrase sequence and has more than 75% identity to SEQ ID NO 10, wherein said identity can be determined using the DNAsis computer program and default parameters;
(m) a nucleic acid sequence which encodes an amino acid sequence which is at least a portion of a plant retroelement reverse transcriptase sequence and has more than 79% identity to SEQ ID NO 12, wherein said identity can be determined using the DNAsis computer program and default parameters;
(n) a nucleic acid sequence which encodes an amino acid sequence which is at least a portion of a plant retroelement protease sequence and has more than 55% identity to SEQ ID NO 14, wherein said identity can be determined using the DNAsis computer program and default parameters;
(o) a nucleic acid sequence which encodes an amino acid sequence which is at least a portion of a plant retroelement RNAseH sequence and has more than 90% identity to SEQ ID NO 16, wherein said identity can be determined using the DNAsis computer program and default parameters;
(p) a nucleic acid sequence which encodes an amino acid sequence which is at least a portion of a plant retroelement sequence and has more than 40% identity to SEQ ID NO 18, wherein said identity can be determined using the DNAsis computer program;
(q) a nucleic acid sequence which encodes an amino acid sequence selected from the group consisting of: SEQ ID NO 4; SEQ ID NO 6; SEQ ID NO 8; SEQ ID NO 10; SEQ ID NO 12; SEQ ID NO 14; SEQ ID NO 16; and SEQ ID NO 18;
(r) a nucleic acid sequence which encodes an allelic variant of an amino acid sequence selected from the group consisting of: SEQ ID NO 4; SEQ ID NO 6; SEQ ID NO 8; SEQ ID NO 10; SEQ ID NO 12; SEQ ID NO 14; SEQ ID NO 16; and SEQ ID NO 18; and
(s) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); a nucleic acid sequence of (c); a nucleic acid sequence of (d); a nucleic acid sequence of (e); a nucleic acid sequence of (f); a nucleic acid sequence of (g); a nucleic acid sequence of (h); a nucleic acid sequence of (i); a nucleic acid sequence of (j); a nucleic acid sequence of (k); a nucleic acid sequence of (l); a nucleic acid sequence of (m); a nucleic acid sequence of (n); a nucleic acid sequence of (o); a nucleic acid sequence of (p); a nucleic acid sequence of (q); and a nucleic acid sequence of (r).
Seeds and plants comprising a nucleic acid as above are particularly provided. Nucleic acid molecules as above which comprise gag, pol and env genes and which comprise adenine-thymidine-guanidine as the gag gene start codon are also particularly provided. Those which comprise gag, pol and env genes, the adenine-thymidine-guanidine as the gag gene start codon, and which further comprises SEQ ID NO 4 are also provided.
Included within the scope of the present invention, with particular regard to the nucleic acids above, are allelic variants, degenerate sequences and homologues. The present invention also includes variants due to laboratory manipulation, such as, but not limited to, variants produced during polymerase chain reaction amplification or site directed mutagenesis. It is also well known that there is a substantial amount of redundancy in the various codons which code for specific amino acids. Therefore, this invention is also directed to those nucleic acid sequences which contain alternative codons which code for the eventual translation of the identical amino acid. Also included within the scope of this invention are mutations either in the nucleic acid sequence or the translated protein which do not substantially alter the ultimate physical properties of the expressed protein. For example, substitution of valine for leucine, arginine for lysine, or asparagine for glutamine may not cause a change in functionality of the polypeptide. Lastly, a nucleic acid sequence homologous to the exemplified nucleic acid molecules (or allelic variants or degenerates thereof) will have at least 85%, preferably 90%, and most preferably 95% sequence identity with a nucleic acid molecule in the sequence listing.
It is known in the art that there are commercially available computer programs for determining the degree of similarity between two nucleic acid sequences. These computer programs include various known methods to determine the percentage identity and the number and length of gaps between hybrid nucleic acid molecules. Preferred methods to determine the percent identity among amino acid sequences and also among nucleic acid sequences include analysis using one or more of the commercially available computer programs designed to compare and analyze nucleic acid or amino acid sequences. These computer programs include, but are not limited to, GCG(trademark) (available from Genetics Computer Group, Madison, Wis.), DNAsis(trademark) (available from Hitachi Software, San Bruno, Calif.) and MacVecto(trademark) (available from the Eastman Kodak Company, New Haven, Conn.). A preferred method to determine percent identity among amino acid sequences and also among nucleic acid sequences includes using the Compare function by maximum matching within the program DNAsis Version 2.1 using default parameters.
Knowing the nucleic acid sequences of the present invention allows one skilled in the art to, for example, (a) make copies of those nucleic acid molecules, (b) obtain nucleic acid molecules including at least a portion of such nucleic acid molecules (e.g., nucleic acid molecules including full-length genes, full-length coding regions, regulatory control sequences, truncated coding regions), and (c) obtain similar nucleic acid molecules from other species. Such nucleic acid molecules can be obtained in a variety of ways including screening appropriate expression libraries with antibodies of the present invention; traditional cloning techniques using oligonucleotide probes of the present invention to screen appropriate libraries of DNA; and PCR amplification of appropriate libraries or DNA using oligonucleotide primers of the present invention. Preferred libraries to screen or from which to amplify nucleic acid molecules include canine cDNA libraries as well as genomic DNA libraries. Similarly, preferred DNA sources to screen or from which to amplify nucleic acid molecules include adult cDNA and genomic DNA. Techniques to clone and amplify genes are disclosed, for example, in Sambrook et al., ibid.
Recombination constructs can be made using the starting materials above or with additional materials, using methods well-known in the art. In general, the sequences can be manipulated to have ligase-compatible ends, and incubated with ligase to generate full constructs. For example, restriction enzymes can be chosen on the basis of their ability to cut at an acceptable site in both sequence to be ligated, or a linker may be added to convert the sequence end(s) to ones that are compatible. The methods for conducting these types of molecular manipulations are well-known in the art, and are described in detail in Sambrook et al., Molecular Cloning. A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) and Ausubel et al., Current Protocols in Molecular Biology (Greene Publishing Associates, Inc., 1993). The methods described herein according to Tinland et al., 91 Proc. Natl. Acad. Sci. USA 8000 (1994) can also be used.
The present invention also includes nucleic acid molecules that are oligonucleotides capable of hybridizing, under stringent hybridization conditions, with complementary regions of other, preferably longer, nucleic acid molecules of the present invention. Oligonucleotides of the present invention can be RNA, DNA, or derivatives of either. The minimum size of such oligonucleotides is the size required for formation of a stable hybrid between an oligonucleotide and a complementary sequence on a nucleic acid molecule of the present invention. Minimal size characteristics are disclosed herein. The present invention includes oligonucleotides that can be used as, for example, probes to identify nucleic acid molecules, primers to produce nucleic acid molecules or therapeutic reagents. Stringent hybridization conditions are determined based on defined physical properties of the gene to which the nucleic acid molecule is being hybridized, and can be defined mathematically. Stringent hybridization conditions are those experimental parameters that allow an individual skilled in the art to identify significant similarities between heterologous nucleic acid molecules. These conditions are well known to those skilled in the art. See, for example, Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, and Meinkoth, et al., 1984, Anal. Biochem. 138, 267-284.
Recombinant molecules of the present invention may also (a) contain secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed protein of the present invention to be secreted from the cell that produces the protein and/or (b) contain fusion sequences which lead to the expression of nucleic acid molecules of the present invention as fusion proteins. Recombinant molecules may also include intervening and/or untranslated sequences surrounding and/or within the nucleic acid sequences of nucleic acid molecules of the present invention.
One embodiment of the present invention includes recombinant vectors, which include at least one isolated nucleic acid molecule of the present invention, inserted into any vector capable of delivering the nucleic acid molecule into a host cell. Such a vector contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to nucleic acid molecules of the present invention and that preferably are derived from a species other than the species from which the nucleic acid molecule(s) are derived. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid. Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulation of nucleic acid molecules of the present invention.
One type of recombinant vector, referred to herein as a recombinant molecule, comprises a nucleic acid molecule of the present invention operatively linked to an expression vector. The phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell. As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified nucleic acid molecule. Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids. Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, endoparasite, insect, other animal, and plant cells.
In particular, expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present invention. In particular, recombinant molecules of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequences that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art. Preferred transcription control sequences include those which function in bacterial, yeast, insect and mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda (such as lambda pL and lambda pR and fusions that include such promoters), bacteriophage T7, T71ac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virus subgenomic promoters), antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such as intermediate early promoters), simian virus 40, retrovirus, actin, retrovirl long terminal repeat, Rous sarcoma virus, heat shock, phosphate and nitrate transcription control sequences as well as other sequences capable of controlling gene expression in prokaryotic or eukaryotic cells. Additional suitable transcription control sequences include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins). Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with plants. The present invention also comprises expression vectors comprising a nucleic acid molecule described herein.
For instance, the following promoters would be useful in early expression of the present sequences: Ogs4B (Tsuchiya et al., 36 Plant Cell Physiology 487 (1994); TA29 (Koltunow et al., 2 Plant Cell 1201 (1990); A3 and A9 (Paul et al., 19 Plant Molecular Biology 611 (1992). In order to then constitutively express the sequences described above, the construct optionally contains, for example, a 35S promoter.
Vectors which comprise the above sequences are within the scope of the present invention, as are plants transformed with the above sequences. Vectors may be obtained from various commercial sources, including Clontech Laboratories, Inc. (Palo Alto, Calif.), Stratagene (La Jolla, Calif.), Invitrogen (Carlsbad, Calif.), New England Biolabs (Beverly, Mass.) and Promega (Madison, Wis.). Preferred vectors are those which are capable of transferring the sequences disclosed herein into plant cells or plant parts.
Recombinant DNA technologies can be used to improve expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications. Recombinant techniques useful for increasing the expression of nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy number plasmids, integration of the nucleic acid molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of nucleic acid molecules of the present invention to correspond to the codon usage of the host cell, deletion of sequences that destabilize transcripts, and use of control signals that temporally separate recombinant cell growth from recombinant enzyme production during fermentation. The activity of an expressed recombinant protein of the present invention may be improved by fragmenting, modifying, or derivatizing nucleic acid molecules encoding such a protein.
Nucleic acids of the present invention may be transferred to cells according to the methods of the present invention, as well as using any of the following well-known means: infective, vector-containing bacterial strains (such as Agrobacterium rhizogenes and Agrobacterium tumefaciens) according to ie. Zambryski, 43 Ann. Rev. Pl. Physiol. Pl. Mol. Biol. 465 (1992); pollen-tube transformation [Zhon-xun et al., 6 Plant Molec. Bio. 165 (1988)]; direct transformation of germinating seeds [Toepfer et al., 1 Plant Cell 133 (1989)]; polyethylene glycol or electroporation transformation [Christou et al., 84 Proc. Nat. Acad. Sci. 3662 (1987)]; and biolistic processes [Yang and Cristou, Particle Bombardment Technology for Gene Transfer (1994)].
The transformed cells may be induced to form transformed plants via organogenesis or embryogenesis, according to the procedures of Dixon Plant Cell Culture: A Practical Approach (IRL Press, Oxford 1987).
Any seed, embryo, plant or plant part is amenable to the present techniques. Of course, the agronomically-significant seeds, embryos, plants or plant parts are preferred. Soybean; maize; sugar cane; beet; tobacco; wheat; barley; poppy; rape; sunflower, alfalfa; sorghum; rose; carnation; gerbera; carrot; tomato; lettuce; chicory; pepper, melon; cabbage; oat; rye; cotton; flax; potato; pine; walnut; citrus (including oranges, grapefruit etc.); hemp; oak; rice; petunia; orchids; Arabidopsis; broccoli; cauliflower; brussel sprouts; onion; garlic; leek; squash; pumpkin; celery; pea; bean (including various legunes); strawberries; grapes; apples; pears; peaches; banana; palm; cocoa; cucumber, pineapple; apricot; plum; sugar beet; lawn grasses; maple; triticale; safflower, peanut; and olive are among the preferred seeds, embryos, plants or plant parts. Particularly preferred are: soybean, tobacco and maize seeds, embryos, plants or plant parts. However, Arabidopsis seeds, embryos, plants or plant parts are also preferred, since it is an excellent system for study of plant genetics.
Preferred are those genes or sequences which are agronomically significant. For example, genes encoding male sterility, foreign organism resistance (viruses or bacteria), including genes which produce bacterial endotoxins, such as bacillus thurigiensis endotoxin, genes involved in specific biosynthetic pathways (eg. in fruit ripening, oil or pigment biosynthesis, seed formation, or carbohydrate metabolism), genes involved in environmental tolerance (eg. salt tolerance, lodging tolerance, cold/frost tolerance, drought tolerance, or tolerance to anaerobic conditions), or genes involved in nutrient content (eg. protein content, carbohydrate content, amino acid content, fatty acid content), genes involved in photosynthetic pathways, or genes involved in self-incompatibility. The choice of gene or sequence induced to recombine in the present invention is not limited. Examples of genes and how to obtain them are available through reference articles, books and supply catalogs, such as The Sourcebook (1-800-551-5291). Sambrook et al., Molecular Cloning. A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) and Weising et al., 22 Ann Rev. Gen. 421 (1988) contain a synthesis of the information that is well-known in this art.
Plant envelope sequences and constructs which comprise the sequences are provided, as are cells, seeds, embryos and plants comprising them. Preferred are isolated nucleic acid molecules, wherein said nucleic acid molecules encode at least a portion of a plant envelope sequence and comprises a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence which has more than 90% identity to SEQ ID NO 5, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which encodes SEQ ID NO 5;
(c) a nucleic acid sequence which encodes an amino acid sequence which has greater than 85% identity to SEQ ID NO 6, wherein said identity can be determined using the DNAsis computer program and default parameters;
(d) a nucleic acid sequence which encodes amino acid sequence SEQ ID NO 6;
(e) a nucleic acid sequence which encodes an allelic variant of SEQ ID NO 6; and
(f) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); a nucleic acid sequence of (c); a nucleic acid sequence of (d); and a nucleic acid sequence of (e).
Plant cells comprising an isolated nucleic acid molecule above are particularly preferred. Also preferred are plant envelope proteins comprising an amino acid sequence encoded by the above. Methods to impart agronomically-significant characteristics to at least one plant cell are also provided, comprising: contacting a plant envelope protein as described to at least one plant cell under conditions sufficient to allow a nucleic acid molecule to enter said cell, wherein said nucleic acid molecule encodes an agronomically-significant characteristic.
Plant integrase sequences and constructs which comprise the sequences are provided, as are cells, seeds, embryos and plants comprising them. Preferred are isolated nucleic acid molecules, wherein said nucleic acid molecules encode at least a portion of a plant integrase sequence and comprises a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence which has more than 90% identity to SEQ ID NO 9, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which encodes SEQ ID NO 9;
(c) a nucleic acid sequence which encodes an amino acid sequence which has greater than 85% identity to SEQ ID NO 10, wherein said identity can be determined using the DNAsis computer program and default parameters;
(d) a nucleic acid sequence which encodes amino acid sequence SEQ ID NO 10;
(e) a nucleic acid sequence which encodes an allelic variant of SEQ ID NO 10; and
(f) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); a nucleic acid sequence of (c); a nucleic acid sequence of (d); and a nucleic acid sequence of (e).
Plant cells comprising an isolated nucleic acid molecule above are particularly preferred. Also preferred are plant integrase proteins comprising an amino acid sequence encoded by the above. Methods to impart agronomically-significant characteristics to at least one plant cell are also provided, comprising: contacting a plant integrase protein as described to at least one plant cell under conditions sufficient to allow a nucleic acid molecule to enter said cell, wherein said nucleic acid molecule encodes an agronomically-significant characteristic.
Plant reverse transcriptase sequences and constructs which comprise the sequences are provided, as are cells, seeds, embryos and plants comprising them. Preferred are isolated nucleic acid molecules, wherein said nucleic acid molecules encode at least a portion of a plant reverse transcriptase sequence and comprises a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence which has more than 90% identity to SEQ ID NO 11, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which encodes SEQ ID NO 11;
(c) a nucleic acid sequence which encodes an amino acid sequence which has greater than 85% identity to SEQ ID NO 12, wherein said identity can be determined using the DNAsis computer program and default parameters;
(d) a nucleic acid sequence which encodes amino acid sequence SEQ ID NO 12;
(e) a nucleic acid sequence which encodes an allelic variant of SEQ ID NO 12; and
(f) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); a nucleic acid sequence of (c); a nucleic acid sequence of (d); and a nucleic acid sequence of (e).
Plant cells comprising an isolated nucleic acid molecule above are particularly preferred. Also preferred are plant reverse transcriptase proteins comprising an amino acid sequence encoded by the above. Methods to impart agronomically-significant characteristics to at least one plant cell are also provided, comprising: contacting a plant reverse transcriptase protein as described to at least one plant cell under conditions sufficient to allow a nucleic acid molecule to enter said cell, wherein said nucleic acid molecule encodes an agronomically-significant characteristic.
Plant RNAseH sequences and constructs which comprise the sequences are provided, as are cells, seeds, embryos and plants comprising them. Preferred are isolated nucleic acid molecules, wherein said nucleic acid molecules encode at least a portion of a plant RNAseH sequence and comprises a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence which has more than 90% identity to SEQ ID NO 15, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which encodes SEQ ID NO 15;
(c) a nucleic acid sequence which encodes an amino acid sequence which has greater than 95% identity to SEQ ID NO 16, wherein said identity can be determined using the DNAsis computer program and default parameters;
(d) a nucleic acid sequence which encodes amino acid sequence SEQ ID NO 16;
(e) a nucleic acid sequence which encodes an allelic variant of SEQ ID NO 16; and
(f) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); a nucleic acid sequence of (c); a nucleic acid sequence of (d); and a nucleic acid sequence of (e).
Plant cells comprising an isolated nucleic acid molecule above are particularly preferred. Also preferred are plant RNAseH proteins comprising an amino acid sequence encoded by the above. Methods to impart agronomically-significant characteristics to at least one plant cell are also provided, comprising: contacting a plant RNAseH protein as described to at least one plant cell under conditions sufficient to allow a nucleic acid molecule to enter said cell, wherein said nucleic acid molecule encodes an agronomically-significant characteristic.
Plant retroelement sequences and constructs which comprise the sequences are provided, as are cells, seeds, embryos and plants comprising them. Preferred are isolated nucleic acid molecules, wherein said nucleic acid molecules encode at least a portion of a plant retroelement sequence and comprises a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence which has more than 95% identity to a nucleic acid sequence selected from the group consisting of: SEQ ID NO 2; SEQ ID NO 5; SEQ ID NO 7; SEQ ID NO 9; SEQ ID NO 11; SEQ ID NO 13; SEQ ID NO 15; and SEQ ID NO 17, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which is selected from the group consisting of: SEQ ID NO 2; SEQ ID NO 5; SEQ ID NO 7; SEQ ID NO 9; SEQ ID NO 11; SEQ ID NO 13; SEQ ID NO 15; and SEQ ID NO 17;
(c) a nucleic acid sequence which encodes an amino acid sequence which has more than 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NO 4; SEQ ID NO 6; SEQ ID NO8; SEQ ID NO 10; SEQ ID NO 12; SEQ ID NO 14; SEQ ID NO 16; SEQ ID NO 18, wherein said identity can be determined using the DNAsis computer program and default parameters;
(d) a nucleic acid sequence which encodes an amino acid sequence selected from the group consisting of: SEQ ID NO 4; SEQ ID NO 6; SEQ ID NO 8; SEQ ID NO 10; SEQ ID NO 12; SEQ ID NO 14; SEQ ID NO 16; and SEQ ID NO 18;
(e) a nucleic acid sequence which encodes an allelic variant of an amino acid sequence selected from the group consisting of: SEQ ID NO 4; SEQ ID NO 6; SEQ ID NO 8; SEQ ID NO 10; SEQ ID NO 12; SEQ ID NO 14; SEQ ID NO 16; and SEQ ID NO18; and
(f) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); a nucleic acid sequence of (c); a nucleic acid sequence of (d); and a nucleic acid sequence of (e).
Nucleic acid molecule as above, which further comprises at least one nucleic acid sequence which encodes at least one agronomically-significant characteristic are preferred. More preferred are those nucleic acid molecules as described wherein the agronomically-significant characteristic is selected from the group consisting of: male sterility; self-incompatibility; foreign organism resistance; improved biosynthetic pathways; environmental tolerance; photosynthetic pathways; and nutrient content. Also more preferred are those isolated nucleic acid molecule as described, wherein the agronomically significant characteristic is selected from the group consisting of: fruit ripening; oil biosynthesis; pigment biosynthesis; seed formation; starch metabolism; salt tolerance; cold/frost tolerance; drought tolerance; tolerance to anaerobic conditions; protein content; carbohydrate content (including sugars and starches); amino acid content; and fatty acid content.
Seeds and plants comprising a nucleic acid molecule as described are also preferred. More preferred are plants as described, wherein the plant is selected from the group consisting of: soybean; maize; sugar cane; beet; tobacco; wheat; barley; poppy; rape; sunflower; alfalfa; sorghum; rose; carnation; gerbera; carrot; tomato; lettuce; chicory; pepper, melon; cabbage; oat; rye; cotton; flax; potato; pine; walnut; citrus (including oranges, grapefruit etc.); hemp; oak; rice; petunia; orchids; Arabidopsis; broccoli; cauliflower, brussel sprouts; onion; garlic; leek; squash; pumpkin; celery; pea; bean (including various legumes); strawberries; grapes; apples; pears; peaches; banana; palm; cocoa; cucumber; pineapple; apricot; plum; sugar beet; lawn grasses; maple; triticale; safflower, peanut; and olive. Most preferred are plants as described which is a soybean plant.
Plant retroelements comprising an amino acid sequence encoded by a nucleic acid sequence described are also provided. Plant cells comprising a nucleic acid molecule described herein, as well as plant retroviral proteins encoded by nucleic acid molecules described herein are provided.
Moreover, methods to transfer nucleic acid into a plant cell, comprising contacting a nucleic acid molecule of the present invention with at least one plant cell under conditions sufficient to allow said nucleic acid molecule to enter at least one cell of said plant are provided. In particular there is provided, methods to impart agronomically-significant characteristics to at least one plant cell, comprising: contacting a plant retroelement of the present invention to at least one plant cell under conditions sufficient to allow a nucleic acid molecule to enter said cell, wherein said nucleic acid molecule encodes an agronomically-significant characteristic. Methods as described, wherein the agronomically-significant characteristic is selected from the group consisting of: male sterility; self-incompatibility; foreign organism resistance; improved biosynthetic pathways; environmental tolerance; photosynthetic pathways; and nutrient content are preferred, as are methods wherein the agronomically-significant characteristic is selected from the group consisting of: fruit ripening; oil biosynthesis; pigment biosynthesis; seed formation; starch metabolism; salt tolerance; cold/frost tolerance; drought tolerance; tolerance to anaerobic conditions; protein content; carbohydrate content (including sugars and starches); amino acid content; and fatty acid content.
Plant retroelement sequences comprising specialized signals, and constructs which comprise the sequences are provided, as are cells, seeds, embryos and plants comprising them. Preferred are isolated nucleic acid molecules, comprising a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence which has more than 95% identity to SEQ ID NO 2; wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which is SEQ ID NO 2;
(c) a nucleic acid sequence which encodes amino acid sequence SEQ ID NO 4; and
(d) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); and a nucleic acid sequence of (c).
Plant retroelements as described above, which further comprise at least one nucleic acid sequence which encodes at least one agronomically-significant characteristic are preferred. More preferred are those methods wherein the agronomically-significant characteristic is selected from the group consisting of: male sterility; self-incompatibility; foreign organism resistance; improved biosynthetic pathways; environmental tolerance; photosynthetic pathways; and nutrient content or those wherein the agronomically significant characteristic is selected from the group consisting of: fruit ripening; oil biosynthesis; pigment biosynthesis; seed formation; starch metabolism; salt tolerance; cold/frost tolerance; drought tolerance; tolerance to anaerobic conditions; protein content; carbohydrate content (including sugars and starches); amino acid content; and fatty acid content.
Preferred are plant retroviral particles comprising an isolated retroelement as described, and seeds and plants comprising the retroelements as described. More preferred plants include soybean; maize; sugar cane; beet; tobacco; wheat; barley; poppy; rape; sunflower; alfalfa; sorghum; rose; carnation; gerbera; carrot; tomato; lettuce; chicory; pepper, melon; cabbage; oat; rye; cotton; flax; potato; pine; walnut; citrus (including oranges, grapefruit etc.); hemp; oak; rice; petunia; orchids; Arabidopsis; broccoli; cauliflower; brussel sprouts; onion; garlic; leek; squash; pumpkin; celery; pea; bean (including various legumes); strawberries; grapes; apples; pears; peaches; banana; palm; cocoa; cucumber; pineapple; apricot; plum; sugar beet; lawn grasses; maple; triticale; safflower; peanut; and olive. Soybean is most preferred.
Also provided are methods to transfer nucleic acid into a plant cell, comprising contacting a plant retroelement as described with at least one plant cell under conditions sufficient to allow said plant retroelement to enter said cell. Methods to impart agronomically-significant characteristics to a plant, comprising contacting a plant retroelement as described with at least one plant cell under conditions sufficient to allow said plant retroelement to enter said cell are also preferred. Those methods wherein the plant retroelement is contacted with said cell via a plant retroviral particle described herein are preferred.
Plant retroviruses are also provided. In particular, plant retroviral particles comprising a plant-derived retrovirus envelope protein are provided. Plant retroviral particles comprising a plant-derived retrovirus envelope protein and which further comprise a plant retroviral protein selected from the group consisting of: plant-derived integrase; plant derived reverse transcriptase; plant-derived gag; and plant-derived RNAseH are preferred.
Plant retroviral particles comprising specialized retroviral proteins, and cells, seeds, embryos and plants which comprise the retroviral particles are provided. Preferred are isolated retroviral particles comprising a plant retroviral protein encoded by a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence comprising (i) a nucleic acid sequence which encodes at least one plant retroviral envelope protein, and (ii) a nucleic acid sequence which has more than 60% identity to a nucleic acid sequence selected from the group consisting of: SEQ ID NO 9; SEQ ID NO 11; SEQ ID NO 15; SEQ ID NO 26; SEQ ID NO 27; SEQ ID NO 28; SEQ ID NO 29; SEQ ID NO 30; and SEQ ID NO 31, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which encodes an amino acid sequence encoded by a nucleic acid sequence (a);
(c) a nucleic acid sequence which encodes an allelic variant of an amino acid sequence encoded by a nucleic acid sequence of (a); and
(d) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); and a nucleic acid sequence of (c).
In particular, there are provided plant retroviral particles, wherein said nucleic acid sequence as described in (a) comprises a plant envelope nucleic acid specifically mentioned in claim 6 is preferred. Those particles which further comprise at least one nucleic acid sequence which encodes at least one agronomically-significant characteristic are preferred.
Also provided are methods to transfer nucleic acid into a plant cell, comprising contacting a plant retroviral particle as described above to at least one plant cell under conditions sufficient to allow said nucleic acid to enter said cell. More preferred are methods to impart agronomically-significant characteristics to a plant, comprising contacting a plant retroviral particle as described to at least one plant cell under conditions sufficient to allow said nucleic acid to enter said cell.
More preferred are isolated retroviral particles comprising a plant retroviral protein encoded by a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence which has more than 80% identity to a nucleic acid sequence selected from the group consisting of: SEQ ID NO 9; SEQ ID NO 11; and SEQ ID NO 15, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which encodes a nucleic acid selected from the group consisting of: SEQ ID NO 9; SEQ ID NO 11; and SEQ ID NO 15;
(c) a nucleic acid sequence which encodes an amino acid sequence encoded by a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); and a nucleic acid sequence of (b);
(d) a nucleic acid sequence which encodes an allelic variant of an amino acid sequence encoded by a nucleic acid selected from the group consisting of: a nucleic acid sequence of (a); and a nucleic acid sequence of (b); and
(e) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); a nucleic acid sequence of (c); and a nucleic acid sequence of (d).
Nucleic acids as above, which further comprises at least one nucleic acid sequence which encodes at least one agronomically-significant characteristic are preferred. More preferred are those nucleic acids wherein the agronomically-significant characteristic is selected from the group consisting of: male sterility; self-incompatibility; foreign organism resistance; improved biosynthetic pathways; environmental tolerance; photosynthetic pathways; and nutrient content, or wherein the agronomically significant characteristic is selected from the group consisting of: fruit ripening; oil biosynthesis; pigment biosynthesis; seed formation; starch metabolism; salt tolerance; cold/frost tolerance; drought tolerance; tolerance to anaerobic conditions; protein content; carbohydrate content (including sugars and starches); amino acid content; and fatty acid content.
Also provided are methods to transfer nucleic acid into a plant cell, comprising contacting a plant retroviral particle as described above to at least one plant cell under conditions sufficient to allow said nucleic acid to enter said cell. More preferred are methods to impart agronomically-significant characteristics to a plant, comprising contacting a plant retroviral particle as described to at least one plant cell under conditions sufficient to allow said nucleic acid to enter said cell.
Also preferred are isolated retroviral particles comprising a plant retroviral protein encoded by a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence which has more than 60% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO 9; SEQ ID NO 11; SEQ ID NO 15; SEQ ID NO 26; SEQ ID NO 27; SEQ ID NO 28; SEQ ID NO 29; SEQ ID NO 30; and SEQ ID NO 31, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which encodes a nucleic acid selected from the group consisting of: SEQ ID NO 9; SEQ ID NO 11; SEQ ID NO 15; SEQ ID NO 26; SEQ ID N027; SEQ ID NO 28; SEQ ID NO 29; SEQ ID NO 30; and SEQ ID NO 31;
(c) a nucleic acid sequence which encodes an amino acid sequence encoded by a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); and a nucleic acid sequence of (b);
(d) a nucleic acid sequence which encodes an allelic variant of an amino acid sequence encoded by a nucleic acid selected from the group consisting of: a nucleic acid sequence of (a); and a nucleic acid sequence of (b); and
(e) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); a nucleic acid sequence of (c); and a nucleic acid sequence of (d).
Also preferred are isolated retroviral particles comprising a plant retroviral sequence encoded by a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence which has more than 80% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO 1; SEQ ID NO 2; SEQ ID NO 3, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which encodes a nucleic acid selected from the group consisting of: SEQ ID NO 1; SEQ ID NO 2; and SEQ ID NO 3;
(c) a nucleic acid sequence which encodes SEQ ID NO 4;
(d) a nucleic acid sequence which encodes an amino acid sequence encoded by a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); and a nucleic acid sequence of (c);
(e) a nucleic acid sequence which encodes an allelic variant of an amino acid sequence encoded by a nucleic acid selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); and a nucleic acid sequence of (c) and
(f) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); a nucleic acid sequence of (c); a nucleic acid sequence of (e); and a nucleic acid sequence of (f).
Plant retroviral particles as described above, which further comprises an envelope-encoding nucleic acid sequence specifically described herein are preferred. Preferred are those retroviral particles which further comprise at least one nucleic acid sequence which encodes at least one agronomically-significant characteristic.
Also provided are methods to transfer nucleic acid into a plant cell, comprising contacting a plant retroviral particle as described above to at least one plant cell under conditions sufficient to allow said nucleic acid to enter said cell. More preferred are methods to impart agronomically-significant characteristics to a plant, comprising contacting a plant retroviral particle as described to at least one plant cell under conditions sufficient to allow said nucleic acid to enter said cell.
Also provided, as part of the present invention, are isolated nucleic acid having at least 20 contiguous nucleotides of the sequence shown in SEQ ID NO 17. xe2x80x9cAt leastxe2x80x9d means that this is the lower limit and the number can be any whole number increment up to the total number of bases in SEQ ID NO 17. For example, isolated nucleic acid sequences which are 25, 30, 35, 40, 45, 50, 55, 60, 65 and 70 are within the scope of the present invention.
The following paragraph is designed to elaborate on the best mode and is not indicative of the sole means for making and carrying out the present invention. This paragraph is not intended to be limiting. The best way to make the present nucleic acids is to clone the nucleic acids from the respective organisms or amplified from genomic cDNA by the polymerase chain reaction using appropriate primers. The best way to make the present retroelements is to assemble the nucleic acids using standard cloning procedures. Transcriptional controls can be manipulated by inserting enhancers in or near the 5xe2x80x2 LTR. Marker genes or genes of interest can be inserted within the retroelement. The best way to make the present retroviral particles is to express the retroelement, preferably at high levels, in plant cells and the particles harvested by sucrose gradient fractionation. The best way to use the present nucleic acids is by allowing retroviral particles to come into contact with plant cells. Expression of marker genes carried by the retroelement can be used as one measure of infection and integration.
Also provided by the present invention are isolated nucleic acid molecules, wherein said nucleic acid molecule encodes at least a portion of a plant retroelement reverse transcriptase and comprises a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence having more than 85% identity to a nucleic acid sequence selected from the group consisting of even-numbered SEQ ID NOs inclusive from SEQ ID NO 42 to SEQ ID NO 164, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which encodes an amino acid sequence having more than 85% identity to an amino acid sequence selected from the group consisting of odd-numbered SEQ ID NOs inclusive from SEQ ID NO 43 through SEQ ID NO 165, wherein said identity can be determined using the DNAsis computer program and default parameters;
(c) a nucleic acid sequence which encodes an allelic variant of a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b).
(d) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b).
Seeds and plants comprising the nucleic acid molecules are also provided, as are nucleic acids as described which comprise gag, pol and env genes and which comprises adenine-thymidine-guanidine as the gag gene start codon. Moreover, those nucleic acids which further comprises SEQ ID NO 5 are also provided. Also provided by the present invention are isolated nucleic acid molecules described, wherein said nucleic acid molecule encodes at least a portion of a plant envelope sequence and comprises a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence which has more than 90% identity to SEQ ID NO 5, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which encodes an amino acid sequence which has greater than 85% identity to SEQ ID NO 6, wherein said identity can be determined using the DNAsis computer program and default parameters;
(c) a nucleic acid sequence which encodes an allelic variant of SEQ ID NO 5; and
(d) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); and a nucleic acid sequence of c).
Plant cells comprising this embodiment are also provided. Methods to impart agronomically-significant characteristics to at least one plant cell, comprising:
contacting a nucleic acid molecule described to at least one plant cell under conditions sufficient to allow at least one agronomically-significant nucleic acid molecule to enter said cell.
Also part of the present invention are isolated nucleic acid molecules, wherein said nucleic acid molecule encodes at least a portion of a plant retroelement reverse transcriptase and comprises a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence having more than 95% identity to a nucleic acid sequence selected from the group consisting of even-numbered SEQ ID NOs inclusive from SEQ ID NO 42 to SEQ ID NO 164, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which encodes an amino acid sequence having more than 95% identity to an amino acid sequence selected from the group consisting of odd-numbered SEQ ID NOs inclusive from SEQ ID NO 43 through SEQ ID NO 165, wherein said identity can be determined using the DNAsis computer program and default parameters;
(c) a nucleic acid sequence which encodes an allelic variant of a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b).
(d) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b).
Seeds and plants comprising the nucleic acid molecules are also provided, as are nucleic acids as described which comprise gag, pol and env genes and which comprises adenine-thymidine-guanidine as the gag gene start codon. Moreover, those nucleic acids which further comprises SEQ ID NO 5 are also provided. Methods to impart agronomically-significant characteristics to at least one plant cell, comprising:
contacting a nucleic acid molecule described to at least one plant cell under conditions sufficient to allow at least one agronomically-significant nucleic acid molecule to enter said cell.
Also provided are isolated nucleic acid molecule, wherein said nucleic acid molecule encodes at least a portion of a plant retroelement reverse transcriptase and comprises a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence selected from the group consisting of even-numbered SEQ ID NOs inclusive from SEQ ID NO 42 to SEQ ID NO 164, wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which encodes an amino acid sequence selected from the group consisting of odd-numbered SEQ ID NOs inclusive from SEQ ID NO 43 through SEQ ID NO 165, wherein said identity can be determined using the DNAsis computer program and default parameters;
(c) a nucleic acid sequence which encodes an allelic variant of a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b).
(d) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b).
Seeds and plants comprising the nucleic acid molecules are also provided, as are nucleic acids as described which comprise gag, pol and env genes and which comprises adenine-thymidine-guanidine as the gag gene start codon. Moreover, those nucleic acids which further comprises SEQ ID NO 5 are also provided. Methods to impart agronomically-significant characteristics to at least one plant cell, comprising:
contacting a nucleic acid molecule described to at least one plant cell under conditions sufficient to allow at least one agronomically-significant nucleic acid molecule to enter said cell.
Nucleic acid molecules of the present invention which further comprise at least one nucleic acid sequence which encodes at least one agronomically-significant characteristic are also provided. Those nucleic acid molecules wherein the agronomically-significant characteristic is selected from the group consisting of: male sterility; self-incompatibility; foreign organism resistance; improved biosynthetic pathways; environmental tolerance; photosynthetic pathways; and nutrient content are preferred. Also preferred are those nucleic acid molecules wherein the agronomically significant characteristic is selected from the group consisting of: fruit ripening; oil biosynthesis; pigment biosynthesis; seed formation; starch metabolism; salt tolerance; cold/frost tolerance; drought tolerance; tolerance to anaerobic conditions; protein content; carbohydrate content (including sugars and starches); amino acid content; and fatty acid content.
Also provided are isolated plant retroviral particles comprising a nucleic acid molecule of the present invention.
Preferred plants are selected from the group consisting of: soybean; maize; sugar cane; beet; tobacco; wheat; barley; poppy; rape; sunflower; alfalfa; sorghum; rose; carnation; gerbera; carrot; tomato; lettuce; chicory; pepper; melon; cabbage; oat; rye; cotton; flax; potato; pine; walnut; citrus (including oranges, grapefruit etc.); hemp; oak; rice; petunia; orchids; Arabidopsis; broccoli; cauliflower, brussel sprouts; onion; garlic; leek; squash; pumpkin; celery; pea; bean (including various legunes); strawberries; grapes; apples; pears; peaches; banana; palm; cocoa; cucumber; pineapple; apricot; plum; sugar beet; lawn grasses; maple; triticale; safflower; peanut; and olive.
In these new aspects of the invention, it is understood that the materials and methods described previously are useful in obtaining the present materials. Moreover, the discussion as to scope and usefulness of the invention, including the percent identities, retroviral uses and constructs, plants transfected, methods for improving crops, etc. are applicable for the present new aspects as well. For instance, combination of the previously disclosed materials with the present materials are certainly within the scope of the present disclosure.